Protein folding is a highly cooperative process; therefore, folding landscapes are usually dominated by the fully folded and the unfolded states (Anfinsen, Science 181: 223-230 (1973); Rose et al., Proc. Natl. Acad. Sci. USA 103: 16623-16633 (2006)). In general, partially unfolded proteins are unstable relative to fully folded proteins and only transiently populated, if at all (Bai et al., Science 269: 192-197 (1995); Bouvignies et al., Nature 477: 111-114 (2011); Hansen et al. J. Biomol. NMR 41: 113-120 (2008); Westerheide and Morimoto, J. Biol. Chem. 280: 33097-33100 (2005); Dul et al., J. Cell Biol. 152: 705-716 (2001)). This suppression of folding intermediates minimizes the availability of aggregation-prone, partially folded species and ensures the success of the folding reaction (Westerheide and Morimoto, J. Biol. Chem. 280: 33097-33100 (2005); Dul et al., J. Cell Biol. 152: 705-716 (2001); Neudecker et al., Science 336: 362-366 (2012); Smith et al., J. Mol. Biol. 330: 943-954 (2003); Chiti et al., EMBO J. 19: 1441-1449 (2000)). Partially unfolded states are of great interest for the insight they contribute into the origins of folding cooperativity (Baldwin, Annu. Rev. Biophys. 37: 1-21 (2008)), folding mechanisms (Englander, Annu. Rev. Bioph. Biom. 29: 213-238 (2000); Sosnick and Barrick, Curr. Opin. Struc. Biol. 21: 12-24 (2011)), functional roles in energy transduction processes (Ihee. et al., Proc. Natl. Acad. Sci. USA 102: 7145-7150 (2005)), and the genesis and propagation of aggregation and misfolding diseases (Neudecker et al., Science 336: 362-366 (2012); Smith et al., J. Mol. Biol. 330: 943-954 (2003); Chiti et al., EMBO J. 19: 1441-1449 (2000)). Direct structural characterization of partially unfolded proteins is challenging because their equilibrium population is usually insignificant (Bouvignies et al., Nature 477: 111-114 (2011)).
Internal ionizable groups are relatively rare. Those that are present invariably play essential roles in energy transduction processes, usually involving H+ or e− transfer reactions (Lanyi, BBA Bioenergetics 1757: 1012-1018 (2006); Pisliakov et al., Proc. Natl. Acad. Sci. USA 105: 7726-7731 (2008); Von Ballmoos et al., Annu. Rev. Biochem. 78: 649-672 (2009)). Internal groups usually titrate with anomalous pKa values because charged species are not compatible with the hydrophobic and dry interior of proteins.
Proteins capable of sensing and responding functionally to small changes in pH near physiological values are of significant biotechnological interest. The structural motif that acts as the pH sensor in naturally occurring pH switch proteins that undergo biologically essential pH-driven conformational transitions usually consists of His residues in interactions with polar or ionizable groups. However, engineering artificial pH sensing proteins by introduction of His residues is challenging. The presently disclosed subject matter is directed to methods for producing proteins comprising artificial pH-sensitive conformational switches within internal regions of the proteins.